The analyses of the biological role of transcription errors will promote our understanding of substrate specificity of RNA polymerase. RNA polymerase errors also have a major impact on transcription pausing and arrest. Better understanding he mechanisms of transcription pausing and errors and their proofreading in living cells will provide additional insights into the side effects of the clinically relevant NTP analogues. We dissected one of the basic mechanisms for transcription pausing involving slow translocation of RNA polymerase along DNA regulated by the length of RNA-DNA hybrid in the elongation complex. This mechanism is shared by prokaryotic and eukaryotic RNA polymerases. Our new findings may be expanded to address the interconnection of transcription elongation, co-transcriptional splicing, and termination.